The use of such chelates has numerous advantages. However, the known reagents and methods have not been as convenient and as effective as is desirable.
In applicant's Canadian patent application 488,513 filed Aug. 12, 1985, (European patent application 85305477.3, filed July 31, 1985) applicant has disclosed assays wherein immunoreactive substances are bound to a solid phase, the substances having connected to them as label or marker a moiety of a ligand which forms a fluorescent chelate with lanthanide metal ion. In order to measure the fluorescence of the chelate, the complex is dissociated with a dissociating solution to cause migration of the ligand from the solid phase to the bulk of the solution. The measurement of the fluorescence in solution results in loss of sensitivity, since the presence of liquid phase causes significant absorption and scattering of the excitation beam and of the emitted light.
Applicant is also aware of prior assay methods in which immunocomplexes are formed bound to a solid phase comprising substances having as marker or label ligands binding lanthanide metal ion, especially europium. In order to determine the quantity of europium which is bound, an enhancement solution is added which releases the europium from the ligand. The solution contains a further ligand which forms a fluorescent chelate with the europium in solution In addition to the disadvantages mentioned above, this method has the drawback that it is vulnerable to europium contamination from the environment of the laboratory or other surroundings, which gives an artificially elevated fluorescence reading in the presence of an excess of the development solution.